mn5y@krebs.acc.Virginia.EDU (Mukund Nori) (09/07/90)
I have been suddenly having the utmost frustrating experiences with ligations.
I am not even able to cut a plasmid and get it to religate to itself. So far,
I have used BRL's T4 ligase and the 5X buffer that comes with it. I have
purified the DNA frm agarose gels using GeneClean, Freeze-squeeze and
electroelution into a trough cut downcurrent of the band and following it into
this selfsame trough. One of my colleagues has even started with triple
banded (on CsCl) material, all to no avail. Yes we have tried fresh enzyme
and all fresh solutions. We even used BRL's supercomp cells to try to get
the presumed-ligated stuff in them. So far, ZILCH!!! Suggestions and ideas
EXTREMELY welcome. Thanks in advance.
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___Raistlin___ Mukund Nori
Raistlin@Virginia.EDU mn5y@krebs.acc.Virginia.EDU
"VIOLENCE IS THE LAST RESORT OF THE INCOMPETENT" Asimov NUM208JN@NRCCAD.NRC.CA (JOHN NASH) (09/08/90)
Hi there,
MN>I have been suddenly having the utmost frustrating experiences with ligations
May I ask a few questions?? I need more information.
1. What is the replicon of the plasmid? Will it replicate in the host you are
transforming? What is the host in question? Which method of transformation
are you attempting?
2. Can you transform uncut plasmid? What is your selection for transformants?
Does the selection require "expression" before plating?
3. What amounts of DNA are you trying to ligate and transform? What
restriction enzyme are you using? Can you detect ligation on a mini-gel?
These questions may seem a bit trivial, but to a reader at the other end of
the message (like me), it makes a difference between speculation and a more
educated guess (is there a difference ;-) ?? )
cheers,
John,
--------------------------------------------------------------
John H.E. Nash <Bitnet: NUM208JN@NRCCAD.NRC.CA >
Institute for Biological Sciences,
National Research Council of Canada,
Ottawa, Canada K1A 0R6.
Phone: (613) 990-0990 Fax: (613) 952-9092.
==============================================================mn5y@krebs.acc.Virginia.EDU (Mukund Nori) (09/11/90)
re: Q1: Yes, the plasmid will grow in the bugs = DH5 alpha from BRL; am
using the Hanahan protocol for transforming them.
re: Q2: Yes, I can transform the uncut plasmid into the bugs; that's how I
made a big plasmid prep. I am using amp plates for selection.
re: Q3: I am using approx 100 ng in 20 ul total reaction volume. Conditions
range from 37 degrees for 4 hours to 15 degrees for 18 hours; using 10 ul
[=0.5 of reaction] to transform 100 ul of bugs and spreading 1/10 onto
amp plates. The plasmids are being cut initially with either BamHI or ClaI.
The DNA is purified from agarose using the freeze-squeeze method and cleaned
using Tris-sat phenol; phenol-chloroform-isoamyl alcohol; chloroform-isoamyl
alcohol; ethanol ppted and resuspended in 30 ul TE.
I understand that these are not quite as trivial as they sound. The same
conditions work like a charm for people across the hall. I am doing a test
using my DNA with all their reagents. Hope to know the answer tomorrow.
I don't know that there is a difference between speculation and educated [:-)]
guesses. Thanks in advance for any suggestions.
******************************************************************
___Raistlin___ Mukund Nori
Raistlin@Virginia.EDU mn5y@krebs.acc.Virginia.EDU
"VIOLENCE IS THE LAST RESORT OF THE INCOMPETENT" Asimov darrin@induction.hsc.usc.edu (Darrin Simmons) (09/12/90)
Does anyone out there have any e. coli delta H1 cells that they could ship me a stab of O/N. I need them for beta-gal fusion protein production with the B-M pEX series expression vector. B-M says they don't ship the cells with the vector and I need them like yesterday. ATCC can ship them to me in 5-14 days but that's a little too late for me. RSVP as soon as possible. Darrin@induction.hsc.usc.edu